To elucidate the effects of ring structure and a substituent on the glutathione peroxidase- (GPx-) like antioxidant activities of aliphatic selenides, series of water-soluble cyclic selenides with variable ring size and polar functional groups were synthesized, and their antioxidant activities were evaluated by NADPH-coupled assay using H2O2 and glutathione (GSH) in water and also by NMR spectroscopy using H2O2 and dithiothreitol (DTT(red)) in methanol. Strong correlations were found among the GPx-like activity in water, the second-order rate constants for the oxidation of the selenides, and the HOMO energy levels calculated in water. The results support the conclusion that the oxidation process is the rate-determining step of the catalytic cycle. On the other hand, such correlations were not obtained for the activity observed in methanol. The optimal ring size was determined to be five. The type of substituent (NH2 < OH < CO2H) and the number can also control the activity, whereas the stereoconfiguration has only marginal effects on the activity in water. In methanol, however, the activity rank could not be explained by the simple scenarios applicable in water.

Interactions of the F 1 F 0 -ATPase subunits between the cytoplasmic domain of the b subunit (residues 26 -156, b cyt ) and other membrane peripheral subunits including ␣, ␤, ␥, ␦, ⑀, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of b cyt fused to the activation domain of the yeast GAL-4, and ␦ subunit fused to the DNA binding domain resulted in the strong expression of the ␤-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of b cyt fused to glutathione S-transferase (GST) together with the ␦ subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-b cyt fusion alone had no such effect, indicating that GST-b cyt was protected by the co-expressed ␦ subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the ␦ subunit in the cell. The affinity purified GST-b cyt did not contain significant amounts of ␦, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified b cyt together with ␣, ␤, ␥, ␦, and ⑀, or with the same combination except ⑀. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and ␦ was stabilized by F 1 subunits other than ⑀ and also suggested that b-␦ interaction was important for F 1 -F 0 interaction.

An engineered Q